The present invention relates to a process for producing polyunsaturated fatty acids in an organism by introducing nucleic acids into said organism which code for polypeptides having acyl-CoA:lysophospholipid-acyltransferase activity. Advantageously, these nucleic acid sequences may, if appropriate together with further nucleic acid sequences coding for biosynthesis polypeptides of the fatty acid or lipid metabolism, be expressed in the transgenic organism.
The invention furthermore relates to the nucleic acid sequences, to nucleic acid constructs comprising the nucleic acid sequences of the invention, to vectors comprising said nucleic acid sequences and/or said nucleic acid constructs and to transgenic organisms comprising the abovementioned nucleic acid sequences, nucleic acid constructs and/or vectors.
A further part of the invention relates to oils, lipids and/or fatty acids produced by the process of the invention and to their use.
Fatty acids and triglycerides have a multiplicity of applications in the food industry, in animal nutrition, in cosmetics and in the pharmacological sector. Depending on whether they are free saturated or unsaturated fatty acids or else triglycerides with an elevated content of saturated or unsaturated fatty acids, they are suitable for very different applications; thus, for example, polyunsaturated fatty acids are added to baby food to improve the nutritional value. Polyunsaturated ω-3-fatty acids and ω-6-fatty acids are, in this connection, an important constituent of animal and human food. Owing to the composition of human food, which is customary today, an addition of polyunsaturated ω-3-fatty acids which are preferably present in fish oils to the food is particularly important. Thus, for example, polyunsaturated fatty acids such as docosahexaenoic acid (=DHA, C22:6Δ4,7,10,13,16,19) or eisosapentaenoic acid (=EPA, C20:5Δ5,8,11,14,17) are added to baby food to improve the nutritional value. The unsaturated fatty acid DHA is said to have a positive effect on brain development.
Hereinbelow, polyunsaturated fatty acids are referred to as PUFA, PUFAs, LCPUFA or LCPUFAs (poly unsaturated fatty acids, PUFA, long chain poly unsaturated fatty acids, LCPUFA).
The various fatty acids and triglycerides are obtained, usually in the form of their triacylglycerides (=triglycerides=triglycerols), mainly from microorganisms such as Mortierella or Schizochytrium or from oil-producing plants such as soybean, oilseed rape, algae such as Crypthecodinium or Phaeodactylum and others. However, they may also be obtained from animals such as, for example, fish. The free fatty acids are advantageously prepared by hydrolysis. Higher polyunsaturated fatty acids such as DHA, EPA, arachidonic acid (=ARA, C20:4Δ5,8,11,14), dihomo-γ-linolenic acid (C20:3Δ8,11,14) or docosapentaenoic acid (DPA, C22:5Δ7,10,13,16,19) cannot be isolated from oil crops, such as oilseed rape, soybean, sunflower, safflower or others. Conventional natural sources of these fatty acids are fish such as herring, salmon, sardine, red fish, eel, carp, trout, halibut, mackerel, zander or tuna, or algae.
Depending on the intended application, preference is given to oils with saturated or unsaturated fatty acids; thus, for example, lipids with unsaturated fatty acids, especially polyunsaturated fatty acids, are preferred in human nutrition. The polyunsaturated ω-3-fatty acids are said to have in this connection a positive effect on the cholesterol level in the blood and thus on the possibility of preventing heart disease. The risk of heart disease, stroke or hypertension may be reduced markedly by adding these ω-3-fatty acids to food. ω-3-fatty acids can also have a positive effect on inflammatory, especially chronically inflammatory, processes in connection with immunological disorders such as rheumatoid arthritis. They are therefore added to food, especially dietetic food, or are applied in medicaments. ω-6-fatty acids such as arachidonic acid tend to have a negative effect on these diseases in connection with said rheumatic disorders, due to our customary foodstuff composition.
ω-3- and ω-6-fatty acids are precursors of tissue hormones, the “eicosanoides, such as the prostaglandins, which are derived from dihomo-γ-linolenic acid, arachidonic acid and eicosapentaenoic acid, the thromoxanes and leukotrienes which are derived from arachidonic acid and eicosapentaenoic acid. Eicosanoides (“PG2 series”) which are formed from ω-6-fatty acids normally promote inflammatory reactions, while eicosanoides (“PG3 series”) from ω-3-fatty acids have little or no proinflammatory effect.
Owing to their positive properties, there has been no lack of attempts in the past to make available genes which are involved in the synthesis of fatty acids or triglycerides for the production of oils in various organisms with a modified content of unsaturated fatty acids. Thus, WO 91/13972 and its US equivalent describe a Δ9-desaturase. WO 93/11245 claims a Δ15-desaturase and WO 94/11516 a Δ12-desaturase. Further desaturases are described, for example, in EP-A-0 550 162, WO 94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-0 794 250, Stukey et al., J. Biol. Chem., 265, 1990: 20144-20149, Wada et al., Nature 347, 1990: 200-203 and Huang et al., Lipids 34, 1999: 649-659. However, the biochemical characterization of the various desaturases has been insufficient to date since the enzymes, being membrane-bound proteins, can be isolated and characterized only with great difficulty (McKeon et al., Methods in Enzymol. 71, 1981: 12141-12147, Wang et al., Plant Physiol. Biochem., 26, 1988: 777-792). Membrane-bound desaturases are normally characterized by being introduced into a suitable organism which is subsequently studied for enzyme activity by analyzing reactants and products. Δ6-desaturases are described in WO 93/06712, U.S. Pat. No. 5,614,393, U.S. Pat. No. 5,614,393, WO 96/21022, WO 00/21557 and WO 99/27111, as is the application for production in transgenic organisms, namely in WO 98/46763, WO 98/46764, WO 9846765. The expression of various desaturases such as those in WO 99/64616 or WO 98/46776 and the formation of polyunsaturated fatty acids are also described and claimed in this connection. Regarding the efficacy of desaturase expression and its influence on the formation of polyunsaturated fatty acids, it should be noted that expression of a single desaturase, as described previously, has resulted in only low contents of unsaturated fatty acids/lipids such as, for example, γ-linolenic acid and stearidonic acid. Furthermore, a mixture of ω-3- and ω-6-fatty acids was usually obtained.
Particularly suitable microorganisms for producing PUFAs are microorganisms such as Thraustochytrium or Schizochytrium strains, algae such as Phaeodactylum tricornutum or Crypthecodinium species, ciliates, such as Stylonychia or Colpidium, fungi such as Mortierella, Entomophthora or Mucor. Strain selection has resulted in the development of a number of mutant strains of the corresponding microorganisms, which produce a series of desirable compounds including PUFAs. However, the mutation and selection of strains with improved production of a particular molecule such as the polyunsaturated fatty acids is a time-consuming and difficult process. Therefore, preference is given, whenever possible, to genetic engineering processes, as described above. However, only limited amounts of the desired polyunsaturated fatty acids such as DPA, EPA or ARA can be produced with the aid of the abovementioned microorganisms, and, depending on the microorganism used, the former are usually obtained as fatty acid mixtures of, for example, EPA, DPA and DHA.
Alternatively, fine chemicals may be produced advantageously on a large scale via production in plants which are developed so as to produce the abovementioned PUFAs. Plants which are particularly well suited for this purpose are oil crops which contain large amounts of lipid compounds, such as oilseed rape, canola, linseed, soybean, sunflower, borage and evening primrose. However, other crop plants containing oils or lipids and fatty acids are also well suited, as mentioned in the detailed description of the present invention. Conventional breeding has been used to develop a number of mutant plants which produce a spectrum of desirable lipids and fatty acids, cofactors and enzymes. However, the selection of new plant cultivars with improved production of a particular molecule is a time-consuming and difficult process or even impossible if the compound does not naturally occur in the respective plant, as is the case with polyunsaturated C18-, C20-fatty acids and C22-fatty acids and those having longer carbon chains.
Owing to the positive properties of unsaturated fatty acids, there has been no lack of attempts in the past to make available these genes which are involved in the synthesis of fatty acids or triglycerides for the production of oils in various plants with a modified content of polyunsaturated fatty acids. Previously, however, it was not possible to produce longer-chain polyunsaturated C20- and/or C22-fatty acids such as EPA or ARA in plants.
However, in other organisms as well as microorganisms such as algae or fungi too, genetically engineered modifications of the fatty acid metabolic pathway via introducing and expressing, for example, desaturases resulted only in relatively small increases in productivity in these organisms. One reason for this may be the high complexity of the fatty acid metabolism. Thus, incorporation of polyunsaturated fatty acids into membrane lipids and/or into triacylglycerides and their degradation and conversion are very complex and, even now, has still not been fully elucidated and understood biochemically and, especially genetically.
The biosynthesis of LCPUFAs and incorporation of LCPUFAs into membranes or triacylglycerides are carried out via various metabolic pathways (Abbadi et al. (2001) European Journal of Lipid Science & Technology 103:106-113). In bacteria such as Vibrio and microalgae such as Schizochytrium, malonyl-CoA is converted via a LCPUFA-producing polyketide synthase to give LCPUFAs (Metz et al. (2001) Science 293: 290-293; WO 00/42195; WO 98/27203; WO 98/55625). In microalgae such as Phaeodactylum and mosses such as Physcomitrelia, unsaturated fatty acids such as linoleic acid or linolenic acid are converted in the form of their acyl-CoAs in multiple desaturation and elongation steps to give LCPUFAs (Zank et al. (2000) Biochemical Society Transactions 28: 654-658). In mammals, the biosynthesis of DHA includes β-oxidation, in addition to desaturation and elongation steps.
In microorganisms and lower plants, LCPUFAs are present either exclusively in the form of membrane lipids, as is the case in Physcomitrella and Phaeodactylum, or in membrane lipids and triacylglycerides, as is the case in Schizochytrium and Mortierella. Incorporation of LCPUFAs into lipids and oils is catalyzed by various acyltransferases and transacylases. These enzymes are already known to carry out the incorporation of saturated and unsaturated fatty acids [Slabas (2001) J. Plant Physiology 158: 505-513; Frentzen (1998) Fett/Lipid 100: 161-166); Cases et al. (1998) Proc. Nat. Acad. Sci. USA 95: 13018-13023]. The acyltransferases are enzymes of the “Kennedy pathway”, which are located on the cytoplasmic side of the membrane system of the endoplasmic reticulum, referred to as “ER” hereinbelow. ER membranes may be isolated experimentally as “microsomal fractions” from various organisms (Knutzon et al. (1995) Plant Physiology 109: 999-1006; Mishra & Kamisaka (2001) Biochemistry 355: 315-322; U.S. Pat. No. 5,968,791). These ER-bound acyltransferases in the microsomal fraction use acyl-CoA as the activated form of fatty acids. Glycerol-3-phosphate acyltransferase, referred to as GPAT hereinbelow, catalyzes the incorporation of acyl groups at the sn-1 position of glycerol 3-phosphate. 1-Acylglycerol-3-phosphate acyltransferase (E.C. 2.3.1.51), also known as lysophosphatidic-acid acyltransferase and referred to as LPAAT hereinbelow, catalyzes the incorporation of acyl groups at the sn-2 position of lysophosphatidic acid, abbreviated as LPA hereinbelow. After dephosphorylation of phosphatidic acid by phosphatidic-acid phosphatase, diacylglycerol acyltransferase, referred to as DAGAT hereinbelow, catalyzes the incorporation of acyl groups at the sn-3 position of diacylglycerols. Apart from these Kennedy pathway enzymes, further enzymes capable of incorporating acyl groups from membrane lipids into triacylglycerides are involved in the incorporation of fatty acids into triacylglycerides, namely phospholipid diacylglycerol acyltransferase, referred to as PDAT hereinbelow, and lysophosphatidylcholine acyltransferase, referred to as LPCAT.
The enzymic activity of an LPCAT was first described in rats [Land (1960) Journal of Biological Chemistry 235: 2233-2237]. A plastic LPCAT isoform [Akermoun et al. (2000) Biochemical Society Transactions 28: 713-715] and an ER-bound isoform [Tumaney and Rajasekharan (1999) Biochimica et Biophysica Acta 1439: 47-56; Fraser and Stobart, Biochemical Society Transactions (2000) 28: 715-7718] exist in plants. LPCAT is involved in the biosynthesis and transacylation of polyunsaturated fatty acids in animals as well as in plants [Stymne and Stobart (1984) Biochem. J. 223: 305-314; Stymne und Stobart (1987) in ‘The Biochemistry of Plants: a Comprehensive Treatise’, Vol. 9 (Stumpf, P. K. ed.) pp. 175-214, Academic Press, New York]. An important function of LPCAT or, more generally, of an acyl-CoA:lysophospholipid acyltransferase, referred to as LPLAT hereinbelow, in the ATP-independent synthesis of acyl-CoA from phospholipids has been described by Yamashita et al. (2001; Journal of Biological Chemistry 276: 26745-26752).
Despite many biochemical data, no genes coding for LPCAT have been identified previously. Genes of various other plant acyltransferases have been isolated and are described in WO 00/18889 (Novel Plant Acyltransferases).
Higher plants comprise polyunsaturated fatty acids such as linoleic acid (C18:2) and linolenic acid (C18:3). Arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are, as described above, found not at all in the seed oil of higher plants, or only in traces (E. Ucciani: Nouveau Dictionnaire des Huiles Végétales. Technique & Documentation—Lavoisier, 1995. ISBN: 2-7430-0009-0). It is advantageous to produce LCPUFAs in higher plants, preferably in oil seeds such as oilseed rape, linseed, sunflower and soybean, since large amounts of high-quality LCPUFAs for the food industry, animal nutrition and pharmaceutical purposes may be obtained at low costs in this way. To this end, it is advantageous to introduce into and express in oil seeds genes coding for enzymes of the biosynthesis of LCPUFAs by genetic engineering methods. Said genes encode, for example, Δ6-desaturase, Δ6-elongase, Δ5-desaturase, Δ5-elongase and Δ4-desaturase. These genes may advantageously be isolated from microorganisms, animals and lower plants which produce LCPUFAs and incorporate them in the membranes or triacylglycerides. Thus, Δ6-desaturase genes have already been isolated from the moss Physcomitrella patens and Δ6-elongase genes have already been isolated from P. patens and the nematode C. elegans. 
First transgenic plants which comprise and express genes coding for enzymes of the LCPUFA biosynthesis and produce LCPUFAs have been described for the first time, for example, in DE 102 19 203 (process for the production of polyunsaturated fatty acids in plants). However, these plants produce LCPUFAs in amounts which require further optimization for processing the oils present in said plants.
In order to enable food and feed to be enriched with these polyunsaturated fatty acids, there is therefore a great need for a simple, inexpensive process for producing said polyunsaturated fatty acids, especially in eukaryotic systems.